CN113061555A

CN113061555A - Screening and application of bacillus strain for producing cellulase

Info

Publication number: CN113061555A
Application number: CN202110425297.6A
Authority: CN
Inventors: 陈修来; 赵阔; 刘立明; 高聪; 刘佳; 郭亮
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-04-20
Filing date: 2021-04-20
Publication date: 2021-07-02
Anticipated expiration: 2041-04-20
Also published as: CN113061555B

Abstract

The invention discloses screening and application of a bacillus strain for producing cellulase, and belongs to the technical field of bioengineering. The bacillus subtilis is screened from animal excrement, the activity of the cellulase produced by fermentation on the shake flask level reaches 118.67U/mL, and the bacillus subtilis has good passage stability. The method for producing the cellulase by using the bacillus subtilis has simple and easy process operation and low culture medium cost, and is suitable for industrial production.

Description

Screening and application of bacillus strain for producing cellulase

Technical Field

The invention relates to screening and application of a bacillus strain for producing cellulase, belonging to the technical field of biological engineering.

Background

The removal of the probiotic properties of feed probiotics also allows the positive effects on the digestive absorption and intestinal health of animals by the production of various chemicals. For example, aiming at diarrhea caused by the fact that animals difficultly absorb crude fibers rich in feed, the feeding probiotics can produce cellulase, the crude fibers which cannot be directly absorbed by the animals are converted into monosaccharide which is easily absorbed and utilized by animal bodies, absorption of the animals is promoted, and the nutritional value of the animals is enriched. Therefore, cellulase has great application in feed.

Compared with the traditional microbial metabolism modification or fermentation optimization method, the method for producing the cellulase by fermenting the feeding probiotics has the advantages that the cost is reduced, the environment is protected, specific substances can obtain higher yield, the probiotics can be well planted in the intestinal tract, no damage can be caused to animal organisms, beneficial substances can be produced, the intestinal flora is enriched, the health of animals is promoted, and the method has important practical significance and application value for modern feed industry.

Aspergillus and Trichoderma are main high-yield strains of cellulase, but the strains need to be modified and optimized by complex genetic process means, such as constitutive overexpression transcription factor, can effectively promote the generation of cellulase and hemicellulase, the enzyme activity can reach 90.38IU/mL, but the cost and the operation difficulty are high, and the production of cellulase is usually inhibited by the growth of thalli, cellulose induction and a carbon source, so that the industrial production of cellulase is greatly inhibited, and the method is not suitable for industrialization. Therefore, researchers turn the research to probiotics with simpler operation so as to achieve high yield of the cellulase by the probiotics. At present, most of bacillus producing cellulase is reported, for example, Lihao and the like separate and screen bacillus subtilis producing cellulase at high yield from humus soil in south China, fermentation conditions of the bacillus subtilis are optimized, and finally the maximum enzyme production level of the bacillus can reach 85.48U/mL after 3 days of fermentation (screening, identification and enzyme production characteristic research of Lihao and other high-yield cellulase strains, Chinese livestock veterinarians 2019,46(03): 711-8). But the fermentation time is still long, and the enzyme production level cannot be well adapted to the industrial production requirement.

Disclosure of Invention

In order to solve the problems of low yield, low production efficiency and large operation difficulty of the existing cellulase, the invention screens a bacillus subtilis strain from animal excrement, can efficiently produce the cellulase and quickly decompose cellulose, and has the probiotic function which is beneficial to the health of animal intestinal tracts.

The first purpose of the invention is to provide a Bacillus subtilis FMME ZK002 for producing cellulase, which is preserved in China center for type culture Collection in 1 month and 29 months in 2021, with the preservation number of CCTCC M2021168, and the preservation address of university of Wuhan, China.

The invention provides a Bacillus subtilis which is preserved in China Center for Type Culture Collection (CCTCC) in 1 month and 29 months in 2021, with the preservation number of CCTCC NO: m2021168.

The invention provides a microbial inoculum, which contains the bacillus subtilis.

In one embodiment, the Bacillus subtilis is present in an amount of not less than 1.0X 10⁶cfu/mL or 1.0X 10⁶cfu/g。

The invention provides a method for producing cellulase, which utilizes bacillus subtilis to produce cellulase.

In one embodiment, Bacillus subtilis cultured to logarithmic growth phase is added to the reaction system, and OD is adjusted₆₀₀0.02 to 0.05.

In one embodiment, the reaction system contains sodium chloride, beef extract and peptone.

In one embodiment, the fermentation system contains 10g/L beef extract, 10g/L peptone and 5g/L sodium chloride.

In one embodiment, the culturing is at 25-35 deg.C, 150-.

In one embodiment, the reaction time is not less than 48 hours.

The invention provides application of the strain in production of cellulase.

The invention provides application of the strain in preparing feed.

The invention has the beneficial effects that: the Bacillus subtilis FMME ZK002 has good passage stability, and the production capacity of cellulase can be stabilized at a certain level after passage; the activity of the cellulase produced by fermentation at the shake flask level reaches 118.67U/mL, and the cellulase has good passage stability. The probiotic bacteria can be well planted in intestinal tracts and promote the health of animals when being applied to the preparation of the feed, so that the probiotic bacteria has important practical significance and application value for the modern feed industry. The method for producing the cellulase by fermentation has simple and easy process operation and low culture medium cost, and is suitable for industrial production.

Biological material preservation

The Bacillus subtilis provided by the invention is classified and named as Bacillus subtilis FMME ZK002, is preserved in China Center for Type Culture Collection (CCTCC) at 1 month and 29 months in 2021, and has a preservation number of CCTCC NO: m2021168, the preservation address is Wuhan university in China.

Drawings

FIG. 1 is a colony morphology of Bacillus subtilis.

FIG. 2 is a growth curve of Bacillus subtilis FMME ZK 002.

FIG. 3 is a graph showing the variation of cellulase activity and cell concentration of Bacillus subtilis in the horizontal shake flask fermentation process.

Detailed Description

(1) Culture medium:

sodium carboxymethylcellulose (CMC-Na) screening medium (g/L): CMC-Na 10, (NH)₄)₂SO₄4, peptone 1, MgSO₄·7H₂O 0.5，KH₂PO₄Sterilizing at 1,121 deg.C for 15 min;

solid activation medium (g/L): 5 parts of sodium chloride, 10 parts of beef extract, 10 parts of peptone, 20 parts of agar and 1.0L of distilled water.

Liquid seed medium (g/L): 5 parts of sodium chloride, 10 parts of beef extract, 10 parts of peptone and 1.0L of distilled water.

Liquid fermentation medium (g/L): 5 parts of sodium chloride, 10 parts of beef extract, 10 parts of peptone and 1.0L of distilled water.

(2) Determination of cellulase:

according to the standard QB 2583-2003, the enzyme activity of the carboxymethyl cellulase is determined by a carboxymethyl cellulase (CMC) enzyme activity determination method. The unit of enzyme activity is specified: under the condition of 60 ℃, the enzyme amount of 1 mu g of glucose is 1 enzyme activity unit when 1mL of cellulase crude enzyme solution hydrolyzes CMC within 1 min.

Pretreatment of fermentation liquor: centrifuging at 4 deg.C and 5000rpm for 10min to obtain supernatant as crude enzyme solution.

Adding 0.4mL of 1% sodium carboxymethylcellulose (CMC-Na) solution into a 25mL test tube, adding 0.4mL of crude enzyme solution, adding no enzyme solution into a blank tube, accurately reacting at 60 ℃ for 30min, taking out, adding 0.5mL of DNS solution, rapidly heating in a 100 ℃ boiling water bath for 10min, rapidly cooling to room temperature, diluting to 5mL, and measuring light absorption value A by using an ultraviolet spectrophotometer₅₄₀And calculating the enzyme activity.

Drawing a cellulase glucose standard curve: 0.5g of analytically pure anhydrous glucose is accurately weighed and prepared into glucose standard solutions of 0.04, 0.06, 0.08, 0.10, 0.12 and 0.14mg/mL by using distilled water. Sucking 2.5mL of each concentration standard solution, placing in 5 test tubes respectively, using water to replace glucose solution as blank control, adding 2.5mL of DNS solution respectively, water bathing at 100 deg.C for 10min, taking out, and rapidly cooling to room temperature with flowing water. Then distilled water was added to the resulting solution to a constant volume of 5mL, and after shaking sufficiently, the absorbance in each tube was measured at a wavelength of 540 nm. And drawing a standard curve by taking the standard glucose concentration of each concentration as an abscissa and taking the light absorption value as an ordinate.

The enzyme activity calculation formula is as follows: x ═ W × N × 1000 × 12.5)/(k × T × M)

(wherein: X, enzyme activity (U/mL); W, absorbance value measured (test group minus blank group); N, dilution factor of enzyme sample where N is 1; kappa; slope of glucose standard curve, 2.9407; T, reaction time where T is 30 min; M, enzyme sample volume where M is 0.4 mL; 1000; mg converted to. mu.g; 12.5 sample dilution factor.)

CMC substrate solution: sodium carboxymethylcellulose (CMC-Na) 1g was weighed and made up to 100mL with phosphate buffer (pH 6). Sterilizing at 121 deg.C for 25min, and storing at 4 deg.C.

Phosphate buffer (pH 6): 21.04g of disodium hydrogen phosphate and 4.26g of sodium dihydrogen phosphate, calibrating the pH value of the solution after the preparation is finished, and finally fixing the volume to 1L.

And (3) detecting crude protein in the feed: see GB/T6432-94 for details.

And (3) determining crude fibers, neutral detergent fibers and acid detergent fibers in the feed: the concrete mode refers to Zhang Chong Yu, Wang Bao Zheng, Zhang Gui, Yi Peng Hui, Lu Shuang Ming, the rapid determination method of the content of crude fiber, NDF, ADF and ADL in the feed [ J ]. Shandong livestock and poultry veterinarian, 2015,36(09):20-22.

Example 1: screening of strains

(1) Screening of Bacillus subtilis for producing cellulase

Primary screening process: taking a proper amount of animal waste samples, dissolving the animal waste samples in sterilized 100mL of distilled water, putting the animal waste samples into a shaking table, shaking the animal waste samples for 30min at the temperature of 30 ℃, and carrying out enrichment culture. Taking the supernatant, diluting by ten times, sucking 10 times^-4、10^-5、10^-6、10^-7、10^-8Respectively coated on CMC-Na screening plates, and cultured in a constant temperature box at 30 ℃ for 24 h. The cellulase-producing strain can generate transparent circles because the cellulase-producing strain does not generate the transparent circles. Therefore, determining the range of potential strains according to whether a cellulose degradation transparent ring exists;

and (3) re-screening: and (3) separating and selecting the strains with potential cellulose degradation capacity by using a basic culture medium. The diameter of the transparent ring is positively correlated with the colony diameter ratio (H/d) to the enzyme-producing activity of the strain. Therefore, the isolated strains were individually spotted onto CMC-Na screening plates, and the ratio of the diameter of the clearing circle to the diameter of the colony was calculated. Selecting the strain with the largest ratio, and preserving at-80 ℃ for later use.

The cellulase-producing strain will produce a transparent ring, while the cellulase-non-producing strain will not produce a transparent ring. A total of 204 colonies were obtained on 5 gradient screening plates, of which the cellulase-producing strain was suspected to be 10. And (3) respectively spotting the 10 strains obtained by primary screening on a CMC-Na screening flat plate, measuring the ratio (H/d value) of the transparent ring/hydrolysis ring to the diameter of the bacterial colony, and performing parallel experiments for 3 times, wherein the H/d value of most strains is lower than 1.5, the H/d value of the strain No. 1 reaches 1.8, and the strain has better cellulose degradation capacity, so that the strain No. 1 is selected for storage.

TABLE 1H/d values of different strains

Strain numbering	1	2	3	4	5	6	7	8	9	10
											H/d value	1.8	1.4	1	1	1.2	1.1	1	1.1	1.2	1.4

(2) Identification of Bacillus subtilis producing cellulase

And (3) strain morphology identification: inoculating the strain obtained by screening on a seed culture medium plate, carrying out inverted culture at the temperature of 30 ℃ for 24h, and observing the growth state of the colony. Meanwhile, a single colony strain is picked by using an inoculating loop, gram staining treatment is carried out, and the microscopic morphology of the strain is observed under an optical microscope.

Strain species identification:

(1) extraction of DNA: inoculating the single colony obtained by screening into a 500mL shake flask containing 50mL seed solution, culturing at 30 ℃ for 12h at 200r/min, carrying out refrigerated centrifugation at 6000rpm for 10min, and collecting thalli. The extraction of total bacterial DNA was carried out exactly as provided by the TaKaRa kit.

(2) Amplification of the 16S rRNA Gene: the upstream primer and the downstream primer of the PCR are respectively universal primers 27F and 5'-AGAGTTTGATCCTGGCTCAG-3' stored in a laboratory; 1492R, 5'-GGTTACCTTGTTACGACTT-3'. The thermal cycle parameters are pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 53 ℃ for 30s, and extension at 72 ℃ for 30s, and after 30 cycles, extension at 72 ℃ for 10 min. The PCR products were sequenced by GENEWIZ Biotech, Suzhou.

(3) Sequence and phylogenetic analysis: the 16S rRNA gene sequences of the strains were subjected to homology analysis by BLAST tool. Multiple sequence alignment the phylogenetic tree (Kimura 2-parameter model) was constructed using the software Clustal 1.83 and finally the maximum similarity method (maximum likehood) and the neighbor-joining method (neighbor-joining) of the Mega 3.1 software.

The bacterial colony and the thallus morphology of the bacterial strain 1 show that the bacterial strain is in a short rod shape, gram staining is positive, spores and periphytic flagella can move, the bacterial colony is circular, the surface is rough and opaque, and the bacterial colony is yellowish and slightly smelly (figure 1).

Extracting the genome of the strain No. 1 according to the instruction of a bacterial genome extraction kit, and carrying out PCR amplification to obtain 16S rRNA of the strain. After obtaining the sequence by sequencing, the sequence was BLAST aligned in NCBI database and the evolutionary tree was constructed using MEGA 7 software. The similarity of the strain 1 and Bacillus subtilis reaches 99.42 percent, the strain 1 is identified as Bacillus subtilis by combining the colony morphological characteristics and 16S rRNA analysis, and the strain is named as Bacillus subtilis FMME ZK 002. Has been preserved in China center for type culture Collection at 29.1.2021, with the preservation address of Wuhan university in China and the preservation number of CCTCC M2021168.

Example 2: bacillus subtilis FMME ZK002 shake flask fermentation

Step 1: preparation of culture Medium

Step 2: seed preparation

And (3) dipping bacterial suspension of the bacterial strain, streaking on a solid culture medium, culturing for about 24 hours in a 30-DEG C constant-temperature incubator to obtain a single bacterial colony, selecting the single bacterial colony, inoculating the single bacterial colony into a 100-mL conical flask containing 25mL of liquid seed culture medium, and culturing for 16 hours (to logarithmic growth phase) in a shaker at 30 ℃ and 200rpm to prepare a seed solution.

And step 3: shake flask fermentation culture

Mixing the obtained seed liquidInoculating into 500mL Erlenmeyer flask containing 100mL liquid fermentation medium according to 1% inoculation amount, and adjusting initial OD of fermentation₆₀₀Is 0.035. The culture was carried out at 30 ℃ for 3 days on a shaker at 200rpm, and the samples were taken once at 12 hours. And centrifuging the fermentation liquor, collecting crude enzyme liquid, and measuring the content of the cellulase in the fermentation liquor. As shown in FIG. 2, the enzyme activities were 9.56U/mL, 35.07U/mL, 87.97U/mL, 106.62U/mL and 110.75U/mL at 0h, 12h, 24h, 36h and 48h of fermentation, respectively, and reached 118.67U/mL at 60h of fermentation (FIG. 3).

Example 3: subculture stability of Bacillus subtilis FMME ZK002

Referring to example 2, the screened Bacillus subtilis FMME 002 was subcultured for 12 generations, and the strain of each generation was inoculated into a fermentation medium for shake flask fermentation. And (3) determining the activity of the cellulase after fermentation is finished, wherein the activity of the cellulase after subculture is shown in table 2, and the activity of the cellulase is maintained at 108-118.67U/mL, so that the Bacillus subtilis FMME ZK002 has high subculture stability.

TABLE 2 cellulase Activity of the passage strains

Example 4: preparation of Bacillus subtilis FMME ZK002 microbial inoculum

Inoculating 300-⁸When the viable count is more than cfu/mL, centrifuging for 10-20 min at 8000-10000 rpm, removing supernatant, sequentially adding buffer solution (normal saline) and cryoprotectant (15-20 g/100mL sucrose solution) in an aseptic environment until the cell concentration is not less than 10⁷And (4) performing vacuum freeze drying treatment when cfu/mL is reached to obtain the microbial agent.

Example 5: application of Bacillus subtilis FMME ZK002 in preparation of feed

Adding the microbial agent prepared from the screened Bacillus subtilis FMME ZK002 into the feed containing the crushed corn flour, corn bran and corn straw cellulose in a mass ratio of 1:2:2, adding water, adjusting the water content to 40%, inoculating the microbial agent prepared in the example 4 into the feed according to an inoculation amount of 0.1% (w/w), fermenting at 35 ℃ for 6-7 days, stopping fermentation when the pH value is 4.5, detecting the measurement of crude protein, crude fiber, neutral detergent fiber and acidic detergent fiber, and finding that the content of the crude protein in the fermented feed is increased by 5.7%, the content of the crude fiber, the neutral detergent fiber and the acidic detergent fiber are respectively reduced by 7.84%, 16.9% and 7.6%, and further detecting that the viable count in the fermented feed reaches 3.6 x 10⁸cfu/mL. Therefore, the feed prepared by using the biological agent of Bacillus subtilis FMME ZK002 to ferment corn processing byproducts as raw materials can effectively reduce the cellulose content in the feed, is beneficial to the digestive absorption of livestock and poultry, and obviously improves the number of live bacteria in the fermented feed.

TABLE 3 Effect of microbial inoculum fermentation on the ingredients in the feed

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A strain of Bacillus subtilis is preserved in China Center for Type Culture Collection (CCTCC) at 29 months 1 in 2021, with the preservation number of CCTCC NO: m2021168.

2. A bacterial preparation comprising the Bacillus subtilis strain of claim 1.

3. The microbial agent according to claim 2, wherein the content of Bacillus subtilis is not less than 1.0X 10⁶cfu/mL or 1.0X 10⁶cfu/g。

4. A method for producing cellulase, characterized in that cellulase is produced by using the Bacillus subtilis of claim 1.

5. The method according to claim 4, wherein Bacillus subtilis cultured to logarithmic growth phase is added to the reaction system and OD is adjusted₆₀₀0.02 to 0.05.

6. The method of claim 5, wherein the reaction system comprises sodium chloride, beef extract and peptone.

7. The method according to claim 6, wherein the culturing is carried out at 25-35 ℃ and 150-250 rpm.

8. The process according to claim 7, wherein the reaction time is not less than 48 h.

9. Use of the strain of claim 1 for the production of cellulase.

10. Use of the strain according to claim 1 for the preparation of feed.